Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma

Vasculogenic mimicry (VM) is the process where cancer cells adopt endothelial characteristics by forming tube-like structures and perfusing channels. This phenomenon has been demonstrated in several types of solid tumors and associated with the growth and survival of tumor cells. In this study, we investigated the presence of VM formation in human pancreatic ductal adenocarcinoma (PDAC) and elucidated the molecular mechanisms underlying the VM process. In human PDAC tissues, CD31-negative, periodic acid-Schiff (PAS)-positive channels were predominantly found in desmoplastic areas, which are generally also hypovascularized. We found a positive correlation of VM capacity to tumor size and NOTCH1 expression and nuclear localization with statistical significance, implicating that Notch activity is involved with VM formation. Additionally, our data showed that the presence of growth or angiogenic factors significantly increased Notch activity in PDAC cell lines and upregulated several mesenchymal marker genes, such as TWIST1 and SNAI1, which can be inhibited by a gamma-secretase inhibitor. Our data showed that Notch signaling plays an important role in inducing VM formation in PDAC by promoting the epithelial-to-mesenchymal transition process.

1. We have confirmed that the manuscript meets PLOS ONE's style requirements as instructed.
2. The additional statement "The written consent was obtained from all participants prior to tissue analysis" has been added to the Tissue Preparation part of the Materials and Methods section on Page 7.
3. On Page 19, we have removed the funding-related information from the manuscript as instructed. The current funding statement is correct. 4. We have included the data that were not previously shown in the manuscript on Pages 16 and 18 as Supplementary Figures 3 and 6, respectively. Additional Responses to the Editor 1. We propose to modify the title of the manuscript to "Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma." We have changed the word "via" to "and" in the title. This modification is to address the comments (point 4) from Reviewer 2. We strongly agree that the role of VE-cadherin should be further explored regarding its role in VM formation. However, it is beyond the scope of our current study at the moment and we are planning to include the function of VE-cadherin, including its expression regulation and activation profiles in our future investigations. We agree with the Reviewer's comment and have included the discussion of VE-cadherin Y658 and Y731 with references in the Conclusion section on Page 17 and as References 26, 27 and 28.
Responses to Comments from Reviewer 2

It is far-fetched that notch1 is localized in the nucleus in the strong VM formation region, and notch1 in the weak VM formation region is localized in the cytoplasm only by IHC experiments (such as fig3A). Could high expression of notch1 in the cytoplasm induce VM production? They
should use more experiments such as IF etc. to further prove this conclusion.
First, while the immunohistochemical analysis of Notch localization in the nucleus is a wellestablished indicator of Notch activity in tissue samples, our results in fact suggested a strong correlation of Notch activity and VM formation in PDAC tissues. In our point of view, Notch immunohistochemistry proved to be a more viable technique than immunofluorescence in order to investigate Notch localization and, at the same time, to identify VM structures, which requires PAS and H&E staining. Second, we also asked a similar question whether VM formation required Notch activity?" This is the reason why we attempted to use an in vitro model to address the question whether Notch signaling plays an essential role in inducing VM structures. Our results indicated that inhibition of Notch signaling in PANC-1 effectively abolished several key characteristics of VM structures, including filopodia formation and cellular branching that were induced by EGF and VEGF. First, the reason why we selected only 20 ng/ml of EGF and VEGF in our study was that the same concentration was also used in endothelial cell culture, making it relevant to study the process that was assumed to replace normal angiogenesis. Moreover, we have tested several concentrations (2, 10, 20, 50 ng/ml) of EGF and VEGF in the past and found that we observed effects of cell morphology change only when we introduced 20 ng/ml or higher of EGF or VEGF, and the higher concentrations did not significantly increase the level of cell morphogical impact. Second, EGF and VEGF treatments increase Notch activity as shown by the upregulation of Notch targets, HEY1 and NRARP. Thus, with this evidence we can infer that Notch is activated and downstream events are induced, in a similar fashion to when Notch is translocated into the nucleus to upregulate target genes.

I think the experimental design of EGF and VEGF promoting cell morphological transformation
is not rigorous enough. If notch1 is knocked out, will the stimulation of PANC-and MIA-PaCa-2 cells by adding EGF and VEGF also promote angiogenesis? Is the strength of promoting angiogenesis the same as the group with normal expression of Notch1?
We asked a very similar question to the reviewer about the importance of NOTCH1 in the setting of EGF and VEGF-induced VM formation. However, we believe that NOTCH1 is also important for other characteristics of PDAC formation and progression. There, we addressed the question by using a well-established gamma-secretase inhibitor DBZ in order to inhibit Notch activation, rather than a complete knockout of NOTCH1. We have shown that DBZ effectively inhibited Notch activity and abolished the effects of EGF-or VEGF-induced cell morphological changes and EMTrelated gene expression induction and added the data in Supplementary Figure 3. Our data provide evidence that EGF and VEGF promote VM formation, which requires Notch activity.

The relevant design of notch1 promoting EMT is not rigorous enough. The experimental results
confirmed that EGF and VEGF promoted EMT, but did not prove the effect of notch. EGF and VEGF promoted EMT and EGF and VEGF can promote the expression of notch1. Didn't let me find the relevant logic.
We made the conclusion about the role of Notch signaling from the data using a gamma-secretase inhibitor, which effectively abolished the effects of EGF and VEGF on the induction of EMT in PDAC. Additionally, there were several previous studies that support the concept of Notch promoting EMT, including in cardiac development and oncogenic transformation (Timmerman LA, et al., 2004), squamous cell carcinoma (Natsuizaka M, et al., 2017), and TGFb-induced retinal fibrosis (Sheng W, et al., 2022). We have added the statement, "Previous studies have demonstrated that Notch signaling plays an important role in activating EMT during normal development and oncogenic transformation" in the Conclusions section on Page 18 (References However, we agree that our data were not strong enough to indicate that EMT was the process that was activated to induce VM formation in PDAC. Therefore, we propose to modify the title of the manuscript to "Notch signaling regulates vasculogenic mimicry and promotes cell morphogenesis and the epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma." We have changed the word "via" to "and" in the title.
Thank you for your kind consideration.